Development of Folate-Group Impedimetric Biosensor Based on Polypyrrole Nanotubes Decorated with Gold Nanoparticles

Abstract

In this study, polypyrrole nanotubes (PPy-NT) and gold nanoparticles (AuNPs) were electrochemically synthesized to form a hybrid material and used as an electroactive layer for the attachment of proteins for the construction of a high-performance biosensor. Besides the enhancement of intrinsic conductivity of the PPy-NT, the AuNPs act as an anchor group for the formation of self-assembly monolayers (SAMs) from the gold-sulfur covalent interaction between gold and Mercaptopropionic acid (MPA). This material was used to evaluate the viability and performance of the platform developed for biosensing, and three different biological approaches were tested: first, the Avidin-HRP/Biotin couple and characterizations were made by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), wherein we detected Biotin in a linear range of 100-900 fmol L^-1. The studies continued with folate group biomolecules, using the folate receptor α (FR-α) as a bioreceptor. Tests with anti-FR antibody detection were performed, and the results obtained indicate a linear range of detection from 0.001 to 6.70 pmol L^-1. The same FR-α receptor was used for Folic Acid detection, and the results showed a limit of detection of 0.030 nmol L^-1 and a limit of quantification of 90 pmol L^-1. The results indicate that the proposed biosensor is sensitive and capable of operating in a range of clinical interests.

Keywords: folate; impedimetric biosensor; modified electrode.

Figure A1

EDS spectra were obtained for the characterization of the electrode modified with PPy (A) and the electrode modified with PPy/AuNPs (B).

Figure A2

Characterization of the PPy/AuNPs-based biosensor construction steps for the Avidin-HRP/Biotin couple. Cyclic voltammograms (A,B) show the characterization of different steps in the construction of the platform-based biosensor, and the Nyquist diagram (C) is for the same characterization.

Figure A3

Adsorption test of different antibody concentrations (0.001, 0.67, 3.30, and 6.60 pmol L⁻¹), using a modified electrode only with PPy. Cyclic voltammograms (A), Nyquist diagram (B).

Figure 1

The SAM formation is due to the covalent interaction between gold and sulfur, which makes possible biomolecule immobilization through the carboxylic groups.

Figure 2

Representative SEM images from the steel mesh coverage: (A,B) closer approximation of a wire mesh, (C) the wire-mesh image of secondary electrons of the hybrid PPy/AuNPs, and (D) the SEM with backscattered electrons. (E,F) TEM representative images from a single nanotube and a small gap in between the steel mash, respectively.

Figure 3

(A) CV for the electrodes modified with just PPy (black) and PPy/AuNPs (red). The Nyquist plot is shown in (B) from electrodes modified with PPy (black) and with PPy/AuNPs (red). The equivalent circuit used to model the EIS results is also inserted as (C).

Figure 4

Cyclic voltammetry (A) and Nyquist plot (B) of the EIS measurement to Biotin detection (100 up to 900 fmol L⁻¹) indicated by colors in both CV and EIS.

Figure 5

Cyclic voltammetry (A) and Nyquist plot (B) to FBP-Ab detection (0.001 up to 6.70 pmol L⁻¹); (C) the EIS response in stability test to 0.001 pmol L⁻¹ of FBP-Ab. The gray measurement was performed in the blank step, while the others correspond to the same antibody concentration.

Figure 6

Folic Acid detection (0.02 up to 113.3 nmol L⁻¹) using the PPy/AuNPs-modified electrode.